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Evaluation of protease-activated receptor 2 in murine models of arthritis.

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Vol. 56, No. 1, January 2007, pp 101–107
DOI 10.1002/art.22312
© 2007, American College of Rheumatology
Evaluation of Protease-Activated Receptor 2 in
Murine Models of Arthritis
Nathalie Busso,1 Matthias Frasnelli,1 Roland Feifel,2 Bruno Cenni,2 Martin Steinhoff,3
Justin Hamilton,4 and Alexander So1
Objective. Protease-activated receptor 2 (PAR-2)
activation has been linked to pro- and antiinflammatory
cellular responses. We undertook this study to explore
the importance of PAR-2 activation in 4 murine models
of arthritis and to analyze the expression of PAR-2 in
human arthritic synovium.
Methods. Zymosan-induced arthritis (ZIA),
K/BxN serum–induced arthritis, and Freund’s complete
adjuvant (CFA)–induced arthritis were generated in
naive PAR-2ⴚ/ⴚ mice and PAR-2ⴙ/ⴙ littermates.
Antigen-induced arthritis (AIA) was generated in immunized mice using methylated bovine serum albumin
(mBSA). The severity of arthritis was assessed by
clinical scoring, technetium uptake measurement, and
histologic analysis. Immune responses to mBSA were
also evaluated from AIA. The expression of PAR-2 in
synovial tissues from rheumatoid arthritis (RA) and
osteoarthritis (OA) patients was compared.
Results. In AIA, arthritis was significantly decreased in PAR-2–deficient mice and was associated
with decreased levels of anti-mBSA IgG antibodies and
lymph node cell proliferation. No difference in arthritis
severity was seen in mice with ZIA, K/BxN serum–
induced arthritis, and CFA-induced arthritis. Synovial
biopsy specimens from RA patients demonstrated significantly increased expression of PAR-2 compared with
those from OA patients.
Conclusion. PAR-2 deficiency was found to modulate articular inflammation in murine models of arthritis that require prior immunization and was associated with reduced levels of anti-mBSA IgG and lymph
node cell proliferation in AIA. Expression of PAR-2 in
RA synovium was significantly higher than that in OA
synovium, and this suggests that PAR-2 is implicated in
the pathogenesis of immune-mediated forms of arthritis.
The protease-activated receptors (PARs) are a
family of transmembrane, G protein–coupled receptors
that are activated by proteolytic cleavage of their extracellular N-terminus (1). Four PARs are currently
known, 3 of which play important roles in the cross-talk
between proteases of the coagulation cascade and cellular activation (2). PAR-2, unlike the other PARs, is
activated by trypsin, mast cell tryptase, and leukocyte
granzymes as well as by the nonthrombin coagulation
proteases tissue factor–factor VIIa complex and factor
Xa, and thus acts as a cellular sensor of inflammation
and coagulation activation. Cleavage of the PAR-2
N-terminus unmasks a tethered ligand sequence (3) that
activates the receptor and downstream signaling pathways, including inositol triphosphate production and
Ca2⫹ mobilization.
Contrasting effects of PAR-2 in inflammation
have been reported (2). PAR-2 may be proinflammatory, since PAR-2 activation increases vascular permeability, neutrophil infiltration (4), and proinflammatory
cytokine secretion and stimulates the release of proin-
Dr. Busso’s work was supported by the Swiss National Scientific Research Fund (grant 3200-067231.01).
Nathalie Busso, PhD, Matthias Frasnelli, MD, Alexander So,
PhD, FRCP: Centre Hospitalier Universitaire Vaudois, Lausanne,
Switzerland; 2Roland Feifel, PhD, Bruno Cenni, PhD: Novartis Institutes for BioMedical Research, Basel, Switzerland; 3Martin Steinhoff,
MD, PhD: University of Münster, Münster, Germany; 4Justin Hamilton, PhD: University of California, San Francisco (current address:
Australian Centre for Blood Diseases, Monash University, Melbourne,
Victoria, Australia).
Drs. Feifel and Cenni own stock and/or hold stock options in
Novartis Pharma. Dr. So has received consulting fees and/or honoraria
(less than $10,000 each) from Abbott Laboratories and Pfizer.
Address correspondence and reprint requests to Alexander
So, PhD, FRCP, Centre Hospitalier Universitaire Vaudois, Service de
Rhumatologie, Avenue Pierre Decker, 1011 Lausanne, Switzerland.
Submitted for publication May 10, 2006; accepted in revised
form September 29, 2006.
flammatory neurogenic peptides from neurons (5). In
murine models of arthritis and multiple sclerosis, PAR2–deficient mice showed a significant reduction of inflammation (6,7). However, antiinflammatory effects of
PAR-2 activation have also been reported in a murine
model of mucosal inflammation (8).
We have previously reported high levels of
thrombin–antithrombin complexes in rheumatoid arthritis (RA) synovial fluid and demonstrated that inhibition
of factor VIIa and thrombin alleviates inflammation in
animal models of arthritis (9,10). We hypothesized that
part of the proinflammatory effects of coagulation proteases in arthritis could be mediated via PAR-2 activation. In order to study the mechanisms and effects of
PAR-2 signaling in arthritis, we analyzed 4 murine
models of arthritis in PAR-2–deficient mice. Our results
indicate that PAR-2 modulates immune-mediated joint
inflammation in the antigen-induced arthritis (AIA)
model, but not in models that did not require prior
immunization. Biopsy specimens from patients showed
that PAR-2 is prominently expressed in RA, but not in
osteoarthritis (OA), synovial tissues, thus reinforcing the
role of PAR-2 signaling in immune-mediated arthritis.
Animal studies. AIA, zymosan-induced arthritis
(ZIA), and Freund’s complete adjuvant (CFA)–induced arthritis were generated in the same PAR-2–deficient mouse strain
(11), whereas K/BxN serum–induced arthritis was generated in
a different strain of PAR-2–deficient animals (12). All experiments used mice that were 8–12 weeks old at the start of the
experiment. Age-matched PAR-2⫹/⫹ littermates were used as
controls. Institutional approval was obtained for all in vivo
Models of experimental arthritis. For AIA, mice were
immunized as described elsewhere (13). Arthritis was induced
on day 21 by intraarticular injection of 100 ␮g of methylated
bovine serum albumin (mBSA) in 10 ␮l of sterile phosphate
buffered saline (PBS) into the right knee; the left knee was
injected with sterile PBS alone. Arthritis was assessed by
measuring technetium uptake as described previously (13).
ZIA was induced by intraarticular injection of 180 ␮g
(6 ␮l) of zymosan A (from Saccharomyces cerevisiae; Sigma, St.
Louis, MO) through the suprapatellar ligament. The contralateral knee was injected with an equal amount of PBS as a
control. Arthritis was assessed by measuring technetium uptake.
For K/BxN serum–induced arthritis, arthritogenic serum was obtained as described elsewhere (14). Arthritis was
induced by intraperitoneal injection of 250 ␮l of K/BxN serum
per recipient mouse on day 0. Arthritis was scored visually in
each paw using a scale of 0 (no signs of inflammation) to 3
(maximal inflammation and swelling), with separate scores for
the proximal and distal joints (maximum score 24). These
assessments were performed by 2 trained individuals who were
blinded to the study group.
CFA-induced arthritis was generated using the protocol described by Ferrell et al (6). Mice were injected in one
knee with CFA (40 ␮l) and in the contralateral knee with PBS.
Arthritis was assessed by measuring technetium uptake.
Histologic grading of arthritis. Tissues and sections
were prepared as described previously (13). Each section was
graded independently by 2 observers who were unaware of the
animal’s genotype. For each histopathologic parameter, the
mean ⫾ SEM score of all slides was calculated.
Humoral and cellular immune response. Measurements
of serum levels of anti-mBSA antibodies and T cell proliferation assays were performed as described previously (15).
Sampling of human tissues. Specimens of synovial
tissue from 9 OA patients and 11 RA patients undergoing
surgery of the knee or hip joint were obtained from the
Department of Orthopedics, Centre Hospitalier Universitaire
Vaudois. Tissues were cut into small pieces, immediately
frozen in precooled hexane, and stored at ⫺70°C until used.
Analyses were performed on consecutive cryostat sections.
PAR-2 immunohistochemistry of human tissues.
Rabbit polyclonal anti–PAR-2 antibody (SC-5597; Santa
Cruz Biotechnology, Santa Cruz, CA) at 10 ␮g/ml final concentration was applied overnight at 4°C on air-dried 5-␮m–
thick cryostat tissue sections. Sections had previously been
fixed for 10 minutes in acetone at 4°C and then incubated for
30 minutes with 10% normal human serum, 10% normal goat
serum, and 1% BSA. Bound primary antibodies were visualized with avidin–biotin–peroxidase complex (Vectastain Elite
ABC kit; Vector, Burlingame, CA). The color was developed
with 3,3⬘-diaminobenzidine (Sigma) containing 0.01% H2O2.
As a control, nonimmune rabbit IgG was used. For doublestaining of RA synovial tissues, mouse monoclonal antibodies
against CD3, CD68, CD20, and vimentin (all from Sigma, Buchs,
Switzerland) were detected by fluorescein isothiocyanate–labeled
anti-mouse antibodies. Rabbit polyclonal anti-human PAR-2
antibodies were detected with rhodamine-labeled anti-rabbit
Reverse transcriptase–polymerase chain reaction
(RT-PCR) for human PAR-2. RNA was extracted from cryostat tissue sections of OA and RA synovial membranes using
TRIzol (Gibco, Basel, Switzerland). RT-PCR was performed
using PAR-2 sense (5⬘-CGTCGGGGCTTCCAGGAG-3⬘) and
antisense (5⬘-GACAGATGCAGAAAACTCATCC-3⬘) primers. As a reference control, GAPDH analysis by RT-PCR was
performed in parallel.
Statistical analysis. Data are reported as the mean ⫾
SEM. The Wilcoxon rank sum test for unpaired variables was
used to compare differences between groups with a nonGaussian distribution. Student’s unpaired t-test was used to
compare groups with normally distributed values. The chisquare statistic was used to compare the frequencies. P values
less than 0.05 were considered significant. All statistical
calculations were performed using the JMP package (JMP
version 4.02; SAS Institute, Cary, NC).
Figure 1. Effects of protease-activated receptor 2 (PAR-2) deficiency in experimental arthritis. Shown is the time course of knee joint inflammation
in PAR-2–deficient mice with antigen-induced arthritis (AIA) (A), zymosan-induced arthritis (ZIA) (B), K/BxN serum–induced arthritis (KIA) (C),
and Freund’s complete adjuvant (CFA)–induced arthritis (CAA) (D). For AIA, ZIA, and CFA-induced arthritis, joint inflammation was measured
by gamma counting of 99mTc uptake on different days after onset of arthritis. Results are expressed as the ratio of 99mTc uptake in the right (R)
arthritic knee joint to that in the left (L) uninflamed knee joint. In A, B, and D, the mean and SEM R:L ratio is shown for each time point. In the
AIA experiment, 22 PAR-2⫹/⫹ mice and 19 PAR-2⫺/⫺ mice were analyzed, whereas 6 mice per genotype were used in the ZIA and CFA-induced
arthritis models. For K/BxN serum–induced arthritis, inflammation of the distal and proximal joints of each paw was visually scored. In C, values
are the mean ⫾ SEM of 8 PAR-2⫹/⫹ mice and 7 PAR-2⫺/⫺ mice. ⴱ ⫽ P ⫽ 0.0031 versus PAR-2⫹/⫹ mice on day 3; ⴱ ⫽ P ⫽ 0.0032 versus PAR-2⫹/⫹
mice on day 7.
Effect of PAR-2 deficiency on experimental arthritis. The role of PAR-2 was tested in 4 experimental
models of arthritis, including AIA and 3 models not
requiring prior immunization (ZIA, K/BxN serum–
induced arthritis, and CFA-induced arthritis). For each
model, PAR-2–deficient mice and their wild-type littermates (PAR-2⫺/⫺ mice and PAR-2⫹/⫹ mice, respectively) were studied. The severity of arthritis was measured by technetium uptake at different time points up
to day 7 after the onset of arthritis for AIA and ZIA and
up to 29 days for CFA-induced arthritis. The severity of
K/BxN serum–induced arthritis was assessed by clinical
scoring. In AIA, technetium uptake (measured as the
mean ⫾ SEM ratio of the uptake in the arthritic knee
joint to the uptake in the uninflamed knee joint) on days
1, 3, and 7 was lower in PAR-2⫺/⫺ mice (n ⫽ 19) than in
PAR-2⫹/⫹ mice (n ⫽ 22) (Figure 1A), with the results
reaching statistical significance on day 3 (1.3 ⫾ 0.05
versus 1.50 ⫾ 0.06; P ⫽ 0.0031) and on day 7 (1.22 ⫾
0.05 versus 1.38 ⫾ 0.05; P ⫽ 0.0032). PAR-2 deficiency
did not attenuate arthritis in the ZIA or CFA-induced
arthritis models (Figures 1B and D) and did not attenuate the clinical severity of arthritis in the K/BxN
serum–induced arthritis model (Figure 1C).
The histologic features of AIA were examined on
day 8 after arthritis onset. Two observers who were
unaware of the animal’s genotype independently graded
Figure 2. PAR-2 effects on immune responses in AIA. Sera were collected on day 8 of AIA. A and B, Levels of anti–methylated bovine serum
albumin (anti-mBSA) IgG antibodies (Ab) (A) and anti-mBSA–specific immunoglobulin isotypes IgG1, IgG2a, and IgG2b (B) were determined by
enzyme-linked immunosorbent assay (ELISA). ⴱ ⫽ P ⬍ 0.02 in A; ⴱ ⫽ P ⫽ 0.036 in B. C, Total levels of immunoglobulin isotypes IgG1, IgG2a, and
IgG2b in serum from nonarthritic, naive PAR-2⫹/⫹ and PAR-2⫺/⫺ mice were determined by ELISA. At least 14 mice per group were used, and
results are expressed as mean and SEM optical density (OD) units. D, Lymph node cell proliferation in response to mBSA (at 0, 1, 10, and 50 ␮g/ml)
was tested in PAR-2⫹/⫹ and PAR-2⫺/⫺ mice by 3H-thymidine incorporation 48 hours after addition of antigen. ⴱⴱ ⫽ P ⬍ 0.006 at 1 ␮g/ml mBSA;
ⴱ ⫽ P ⫽ 0.007 at 10 ␮g/ml mBSA. aBSA ⫽ anti-mBSA (see Figure 1 for other definitions).
synovial thickness and cartilage damage. The mean ⫾
SEM synovial thickness score on day 8 of arthritis was
significantly attenuated in PAR-2⫺/⫺ mice (n ⫽ 20)
compared with PAR-2⫹/⫹ mice (n ⫽ 22) (3.41 ⫾ 0.3
versus 4.76 ⫾ 0.24; P ⬍ 0.01) (results not shown).
Mean ⫾ SEM cartilage damage scores were slightly,
but not significantly, decreased in PAR-2⫺/⫺ mice
(n ⫽ 20) compared with PAR-2⫹/⫹ mice (n ⫽ 22)
(2.6 ⫾ 0.3 versus 3.26 ⫾ 0.19; P ⫽ 0.17) (results not
shown). In ZIA and CFA-induced arthritis, there were
no differences in histologic scoring, either for synovial
thickness or cartilage damage, between wild-type and
PAR-2–deficient mice (results not shown). Histologic
analysis was not performed in mice with K/BxN
serum–induced arthritis.
Effect of PAR-2 deficiency on immune responses
in AIA. We examined the immune responses to mBSA in
AIA. Anti-mBSA antibodies were significantly lower in
arthritic PAR-2⫺/⫺ mice (n ⫽ 14) than in PAR-2⫹/⫹
mice (n ⫽ 22) (mean ⫾ SEM 0.55 ⫾ 0.09 optical density
[OD] units versus 1.11 ⫾ 0.15 OD units; P ⬍ 0.02)
(Figure 2A). Total serum levels of IgG isotypes were
identical in the 2 strains (Figure 2C), but levels of
anti-mBSA IgG2b were significantly lower in PAR-2⫺/⫺
mice (Figure 2B).
The role of PAR-2 in T cell immune responses
was also examined. Lymphocytes from draining lymph
nodes were stimulated in vitro with mBSA. 3Hthymidine uptake was significantly decreased in cells
isolated from PAR-2⫺/⫺ mice compared with cells
isolated from PAR-2⫹/⫹ mice (P ⬍ 0.006 and P ⫽
0.007 at 1 and 10 ␮g/ml mBSA, respectively) (Figure
Expression of PAR-2 in RA synovial membranes.
PAR-2 expression was determined in synovial biopsy
specimens from RA and OA patients was compared by
RT-PCR (Figure 3A) and by immunohistology (Figure
3B). In RA synovial tissues, 5 of 11 specimens expressed
PAR-2 by RT-PCR, whereas it was not detected in any
of the 9 specimens from OA patients (␹2 ⫽ 7.33, P ⫽
0.007). The differential expression of PAR-2 in RA
Figure 3. Protease-activated receptor 2 (PAR-2) expression in human synovial tissues. A, Synovial mRNA from osteoarthritis (OA) patients (lane
3) and rheumatoid arthritis (RA) patients (lane 4) was analyzed by reverse transcriptase–polymerase chain reaction (PCR) using PAR-2 primers.
PCR with GAPDH primers was performed as a control for RNA quality. No RNA was added to the reaction in lane 1 (negative control). Gut mRNA
was used as a positive control (lane 2). B, PAR-2 immunohistochemistry in OA and RA synovial membranes. In RA tissues, the lining (d) and
sublining (e and f) layers showed faint-to-moderate staining. Within the inflammatory infiltrate, some cells were positive. In contrast, no PAR-2
staining was detected in OA tissues (a–c). Negative controls on RA tissues, using nonimmune rabbit serum instead of primary antibody to rabbit
PAR-2, showed no staining (g and h). C, Characterization of PAR-2–expressing cells within the RA synovial membrane. Staining for mouse
anti-human CD3, CD68, vimentin, and CD20 was detected with fluorescein isothiocyanate–labeled anti-mouse antibodies (green fluorescence).
Rabbit polyclonal anti-human PAR-2 antibodies were detected with anti-rabbit antibodies conjugated with rhodamine (red fluorescence).
Double-staining immunofluorescence analysis demonstrated PAR-2 expression in some fibroblasts, a subset of macrophages, and T and B cells.
tissues versus OA tissues was confirmed by immunohistochemistry. In RA tissues, lining and sublining synovial
cells showed faint-to-moderate staining (Figure 3, parts
d–f). Within the RA synovium, positive staining was also
seen around some inflammatory aggregates. The vascular endothelium and/or smooth muscle was faintly posi-
tive in some vessels. In contrast, PAR-2 staining was
absent in the 9 OA tissues examined (Figure 3B, parts
a–c). Double-staining immunofluorescence with cellspecific markers (Figure 3C) demonstrated PAR-2 expression in a subset of vimentin-positive cells, CD68⫹
cells, and T and B cells.
PAR-2 is capable of modulating inflammation by
multiple mechanisms (2), but its role in arthritis has not
been clearly established. Mast cell tryptase can activate
PAR-2, and increased numbers of mast cells are present
in synovial tissues from patients with RA. Although
thrombin itself does not activate PAR-2, it is capable of
amplifying the generation of upstream proteases (tissue
factor–factor VIIa complex, factor Xa, and the cognate
ternary complex) implicated in PAR-2 cleavage and
could be one reason why the inhibitor hirudin is capable of reducing the severity of collagen-induced arthritis (10).
Based on the reported data, we expected that
PAR-2 deficiency would abrogate joint inflammation.
Unexpectedly, we found that PAR-2 deficiency attenuated joint inflammation only in the AIA model and not
in the ZIA, CFA-induced arthritis, and K/BxN serum–
induced arthritis models. The main difference is the
requirement of prior immunization in AIA, which is not
needed in the other 3 models. We therefore investigated
immune responses to mBSA in the context of PAR-2
deficiency, and we observed a marked reduction in
humoral and cellular responses to mBSA. These findings
suggest that PAR-2 is capable of modulating antigenspecific immune responses but that it plays a negligible
role in “innate immune” pathways of arthritis.
Our results differ from those obtained in a study
of CFA-induced monarthritis, an unusual model of
murine arthritis, which showed that PAR-2 deficiency
virtually abolished inflammatory arthritis (6). We have
tried to duplicate the reported results without success. In
our experience, arthritis induced in this manner did not
differ between PAR-2⫺/⫺ and PAR-2⫹/⫹ animals, and
histologic scores were equivalent (data not shown).
Possible explanations for the discrepant results include
differences in the genetic background of the deficient
mice or in the gene construct of the PAR-2 deletion.
Throughout our experiments, we used wild-type littermates as controls for the deficient animals in order to
minimize the influence of background genetic and environmental factors.
Using immunohistochemistry, we showed that
PAR-2 was expressed in RA synovial tissue, particularly
in the lining layer and interstitial tissues and in a subset
of fibroblasts and immune cells within the synovium. In
contrast, no PAR-2 expression was seen in OA synovium. It appears that PAR-2 expression is not limited to
a single cell type, as demonstrated by double-staining
immunofluorescence. However, it is present on only a
subset of macrophages and T and B cells. At present, we
have no indication of what restricts expression to these
subsets. Unfortunately, the available antibodies against
murine PAR-2 were unsuitable for immunohistochemistry, so we are not able to provide details of its
distribution in murine arthritis.
In conclusion, our data show that PAR-2 plays a
role in immune-mediated joint inflammation but not in
innate immune models of joint inflammation. The decreased severity of AIA in mice with PAR-2 deficiency
and the prominent expression of PAR-2 in RA synovium
suggest that PAR-2 activation is implicated in immune
inflammatory diseases such as RA and may be an
interesting target for future intervention studies.
We thank Carole Herkenne-Morard and Véronique
Chobaz for excellent technical assistance and Eric Kolo for the
confocal microscopy analysis.
Dr. So had full access to all of the data in the study and takes
responsibility for the integrity of the data and the accuracy of the data
Study design. Drs. Busso, Cenni, Steinhoff, and So.
Acquisition of data. Drs. Busso, Frasnelli, Feifel, Cenni, and Hamilton.
Analysis and interpretation of data. Drs. Busso, Frasnelli, Feifel,
Cenni, Steinhoff, and So.
Manuscript preparation. Drs. Busso, Cenni, Steinhoff, Hamilton, and So.
Statistical analysis. Drs. Busso and Feifel.
Provision of critical reagents. Dr. Hamilton.
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